Process for making beta 3 agonists and intermediates

ABSTRACT

The present invention is directed to processes for preparing beta 3 agonists of Formula (I) and Formula (II) and their intermediates. The beta 3 agonists are useful in the treatment of certain disorders, including overactive bladder, urinary incontinence, and urinary urgency.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. application Ser. No.14/354,158, filed on Apr. 25, 2014, which is a 371 National StageApplication of PCT/US12/61252, filed on Oct. 22, 2012, which claimspriority from U.S. Provisional Application No. 61/552,195 filed on Oct.27, 2011.

BACKGROUND OF THE INVENTION

This application is directed to synthetic processes for making beta 3agonists of Formula (I) and Formula (II) and their intermediatecompounds.

Beta Adrenergic receptors (βAR) are present in detrusor smooth muscle ofvarious species, including human, rat, guinea pig, rabbit, ferret, dog,cat, pig and non-human primate. However, pharmacological studiesindicate there are marked species differences in the receptor subtypesmediating relaxation of the isolated detrusor; β1AR predominate in catsand guinea pig, β2AR predominate in rabbit, and β3AR contribute orpredominate in dog, rat, ferret, pig, cynomolgus and human detrusor.Expression of βAR subtypes in the human and rat detrusor has beenexamined by a variety of techniques, and the presence of β3AR wasconfirmed using in situ hybridization and/or reversetranscription-polymerase chain reaction (RT-PCR). Real time quantitativePCR analyses of β1AR, β2AR and β3AR mRNAs in bladder tissue frompatients undergoing radical cystectomy revealed a preponderance of PARmRNA (97%, cf 1.5% for β1AR mRNA and 1.4% for β2AR mRNA). Moreover, β3ARmRNA expression was equivalent in control and obstructed human bladders.These data suggest that bladder outlet obstruction does not result indownregulation of β3AR, or in alteration of PAR-mediated detrusorrelaxation. β3AR responsiveness also has been compared in bladder stripsobtained during cystectomy or enterocystoplasty from patients judged tohave normal bladder function, and from patients with detrusorhyporeflexia or hyperreflexia. No differences in the extent or potencyof β3AR agonist mediated relaxation were observed, consistent with theconcept that the β3AR activation is an effective way of relaxing thedetrusor in normal and pathogenic states. Functional evidence in supportof an important role for the β3AR in urine storage emanates from studiesin vivo. Following intravenous administration to rats, the rodentselective β3AR agonist CL316243 reduces bladder pressure and incystomeric studies increases bladder capacity leading to prolongation ofmicturition interval without increasing residual urine volume.

Overactive bladder (OAB) is characterized by the symptoms of urinaryurgency, with or without urgency urinary incontinence, usuallyassociated with frequency and nocturia. The prevalence of OAB in theUnited States and Europe has been estimated at 16 to 17% in both womenand men over the age of 18 years. Overactive bladder is most oftenclassified as idiopathic, but can also be secondary to neurologicalcondition, bladder outlet obstruction, and other causes. From apathophysiologic perspective, the overactive bladder symptom complex,especially when associated with urge incontinence, is suggestive ofdetrusor overactivity. Urgency with or without incontinence has beenshown to negatively impact both social and medical well-being, andrepresents a significant burden in terms of annual direct and indirecthealthcare expenditures. Importantly, current medical therapy forurgency (with or without incontinence) is suboptimal, as many patientseither do not demonstrate an adequate response to current treatments,and/or are unable to tolerate current treatments (for example, dry mouthassociated with anticholinergic therapy).

The present invention describes efficient and economical processes asdescribed in more detail below for the preparation of the beta 3agonists of Formula (I) and Formula (II) and intermediate compounds thatcan be used for making these agonists.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (FIG. 1) is a powder X-ray diffraction pattern of the crystallineanhydrous form of compound i-11 of Example 1.

FIG. 2 (FIG. 2) is a powder X-ray diffraction pattern of the crystallinehemihydrate form of compound i-11 of Example 1.

SUMMARY OF THE INVENTION

The present invention is directed to synthetic processes for making beta3 agonists of Formula (I) and Formula (II) and their intermediatecompounds I-11 and I-12.

DESCRIPTION OF THE INVENTION

Described herein is a process of making compound I-11 from compound I-5bthrough multiple step reactions:

In one embodiment, the multiple-step reactions from compound I-5b tocompound I-11 comprise reacting compound I-5b with acetone and P¹ ₂O toproduce compound I-6:

wherein P¹ is selected from the group consisting of Ac, Bn, Boc, Bz,Cbz, DMPM, FMOC, Ns, Moz, and Ts. In one embodiment, P¹ is Boc.

In one embodiment, the multiple-step reactions from compound I-5b tocompound I-11 comprise oxidizing compound I-6 with an oxidizing agent inthe presence of a catalyst to produce compound I-7:

wherein P¹ is selected from the group consisting of Ac, Bn, Boc, Bz,Cbz, DMPM, FMOC, Ns, Moz, and Ts. In one embodiment, P¹ is Boc.

Suitable oxidizing agents include, but are not limited to, NaOCl,NaClO₂, hydrogen peroxide, Swern oxidation and variants such as pyridinesulfur trioxide, PCC, and DCC. In one embodiment, the oxidizing agent isNaOCl.

The amount of the oxidizing agent is typically 1.1 equiv. to 1.3 equiv.,or more specifically, 1.2 equiv. to 1.25 equiv. In one embodiment, theamount of the oxidizing agent is 1.25 equiv.

Suitable catalysts for the above oxidation reaction include, but are notlimited to, TEMPO and TEMPO analogues. In one embodiment, the catalystis TEMPO.

One advantage of the presently described process is that compound I-7from the oxidation step can be used directly in the next Homer WadsworthEmmons (hereinafter, “HWE”) step to make compound I-8. This one potprocess eliminates the need for solvent switch and can increase theyield and reduce cost.

In one embodiment, the oxidation step from I-6 to I-7 can be carried outin the presence of a solvent. Suitable solvents include, but are notlimited to, THF, MTBE, CH₂Cl₂, MeCN, toluene and mixtures thereof. Inone embodiment, the solvent is a mixture of toluene and MeCN. In anotherembodiment, the solvent is a mixture of CH₂Cl₂ and MeCN.

In one embodiment, the multiple-step reactions from compound I-5b tocompound I-11 comprise reacting compound I-7 with phosphate compoundA-4:

in the presence of a solvent to produce compound I-8 (“HWR reaction”):

wherein P¹ and P² are each independently selected from the groupconsisting of Ac, Bn, Boc, Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts. In oneembodiment, both P¹ and P² are Boc.

Suitable solvents include, but are not limited to, THF, MTBE, CH₂Cl₂,MeCN, toluene and a mixture comprising two of the foregoing solvents. Inone embodiment, the solvent is the mixture of toluene and MeCN.

The HWE reaction is typically carried out at a temperature of −10 to 50°C., or more specifically, 0 to 40° C. In one embodiment, the temperatureis 0 to 25° C. In another embodiment, the temperature is 40° C.

The HWE reaction is typically carried out in the presence of a base or asalt. In one embodiment, the base is a tertiary amine. In anotherembodiment, the base is N,N-diisopropylethylamine (DIPEA).

In one embodiment, the salt is lithium halide, or more specifically,LiCl or LiBr.

In the HWE reaction, an impurity compound I-21 (aldol dimmer by-product)may be formed in addition to compound I-8:

It has been found that by adjusting pH to between 6.5 and 7.0 after thereaction, higher purity compound I-8 can be obtained with improvedyield. Additionally, addition of more reactant compound A-4 has beenshown to drive the impurity I-21 to product I-8. In one embodiment,addition of an extra 0.2 equiv. of A-4 can reduce the level of I-21 tofrom 8 LCAP to 2 LCAP.

Increasing the reaction temperature can speed up the conversion to thedesired product compound I-8 and reduce the level of the byproductcompound I-21.

By changing the reaction from a batch process to an addition controlledprocess, the yield of compound I-8 can be improved and the level ofbyproduct compound I-21 can be reduced. For example, by adding reactantcompound I-7 to a solution containing reactant compound A-4, the levelof I-21 can be decreased and the yield of compound I-8 improved.

In one embodiment, a solution containing 1.2 equiv of A-4, 3 equiv. ofDIPEA and 3 equiv. of LiCl in 5 volumes of MeCN was prepared and warmedto 40° C. A toluene stream of compound I-7 was then added to thismixture over 3 h, after an additional 30 min aging conversion to productwas complete. The level of impurity I-21 was about ˜1 LCAP. Sampling thereaction at 1 h intervals showed there was no build-up of compound I-7in the reaction mixture. After work up the product was isolated with a90% isolated yield.

It has also been found that using slightly smaller amount of reactantA-4 does not negatively affect the yield of compound I-8. In oneembodiment, 1.0 instead of 1.2 equiv. of compound A-4 was used and highyield was still obtained.

Compound A-4 used in the HWE reaction can be prepared from compound A-1:

using similar synthetic steps and conditions as described in A GeneralProcedure for the Preparation of β-Ketophosphonates, Maloney et. al., J.Org. Chem., 74, page 7574-7576 (2009).

In one embodiment, the reduction of compound I-8 to produce compound I-9is carried out in the presence of a catalyst:

wherein P¹ and P² are each independently selected from the groupconsisting of Ac, Bn, Boc, Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts. In oneembodiment, both P¹ and P² are Boc.

Suitable catalysts include, but are not limited to, Pd, Raney Ni, Pt,PdCl₂, and Pd(OH)₂. In one embodiment, the catalyst is 5% Pd/C.

In another embodiment, the reduction from I-8 to I-9 is carried out inthe presence of a solvent. Suitable solvents include, but are notlimited to, THF, MTBE, CH₂Cl₂, MeCN, toluene, methanol, ethanol,2-propanol and mixtures thereof. In one embodiment, the solvent is THF.

In another embodiment, the reduction reaction is carried out usinghydrogen gas at a pressure of 2 to 300 psig, preferably about 40 psig,in the presence of a catalyst.

In one embodiment, compound I-9 reacts with an acid to produce compoundI-10 through a cyclization reaction:

Suitable acids include, but are not limited to, HCl, HBr, TFA, MeSO₃H,TfOH, H₂SO₄, para-toluenesulfonic acid, and other sulfone acids such asRSO₃H wherein R is C₁₋₆alkyl, aryl or substituted aryl. In oneembodiment, the acid is HCl.

In one embodiment, HCl is used as acid and an HCl salt of compound I-10is obtained. In one embodiment, the HCl salt is in the form of bis-HClsalt. In another embodiment, the bis-HCl salt is in the form of amono-hydrate. In another embodiment, the mono-hydrate of the bis-HClsalt of compound I-10 is a crystalline material.

The conversion from I-9 to I-10 can be carried out at a temperature of 0to 40° C., or more specifically, 15 to 25° C., or even morespecifically, 20 to 25° C. In one embodiment, the temperature is 20 to25° C.

In one embodiment, compound I-10 is reduced to compound I-11 in thepresence of a catalyst:

The reaction conditions for the conversion from I-10 to I-11 can becontrolled so a cis-selective hydrogenation process is obtained. In oneembodiment, the cis-selective hydrogenation is carried out in thepresence of a catalyst. Suitable catalysts include, but are not limitedto Pt on alumina, Pd on alumina, Pd/C, Pd(OH)₂—C, Raney Ni, Rh/C, Rh/Al,Pt/C, Ru/C and PtO₂. In one embodiment, the catalyst is Pt on alumina.

In another embodiment, the cis-selective hydrogenation from I-10 to I-11is carried out in the presence of HMDS, which can protect the hydroxygroup in situ and therefore improve the diastereo selectivity. Othersuitable protecting reagents include, but are not limited to, TMSCl,TESCl, and TBDMSCl.

In one embodiment, compound I-11 is obtained in the form of acrystalline anhydrous free base. In another embodiment, compound I-11 isobtained in the form of a crystalline free base hemihydrate.

In one embodiment, a process of making compound I-11 comprises:

(a) reducing compound I-8:

in the presence of a catalyst to produce compound I-9:

(b) reacting compound I-9 with an acid to produce compound I-10:

and

(c) reducing compound I-10 in the presence of a catalyst to producecompound I-11:

wherein P¹ and P² are each independently selected from the groupconsisting of Ac, Bn, Boc, Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts.

In one embodiment, the catalyst in step (a) above is selected from thegroup consisting of Pd, Raney Ni, Pt, PdCl₂, and Pd(OH)₂.

In one embodiment, the acid in step (b) above is selected from the groupconsisting of HCl, HBr, TFA, MeSO₃H, TfOH, H₂SO₄, para-toluenesulfonicacid, and RSO₃H wherein R is alkyl, aryl or substituted aryl.

In one embodiment, the reduction of step (c) is carried out in thepresence of HMDS and the catalyst used is selected from the groupconsisting of Pt on alumina, Pd on alumina, Pd/C, Pd(OH)₂—C, Raney Ni,Rh/C, Rh/Al, Pt/C, Ru/C and PtO₂.

In one embodiment, a process of making compound I-11 comprises:

(a) reacting compound I-7:

with phosphate compound A-4:

to produce compound I-8:

wherein the reaction is carried out at a temperature of about 20 to 40°C. and in the presence of a solvent selected from the group consistingof THF, MTBE, CH₂Cl₂, MeCN, toluene and a mixture comprising two of theforegoing solvents;

(b) reducing compound I-8 in the presence of a catalyst selected fromthe group consisting of Pd, Raney Ni, Pt, PdCl₂, and Pd(OH)₂ to producecompound I-9:

(c) reacting compound I-9 with an acid to produce compound I-10:

and

(d) reducing compound I-10 in the presence of a catalyst to producecompound I-11:

wherein P¹ and P² are each independently selected from the groupconsisting of Ac, Bn, Boc, Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts.

In another embodiment, a process of making compound I-11 comprises:

(a) reacting compound I-5b:

with acetone and Boc₂O to produce compound I-6:

(b) oxidizing compound I-6 with an oxidizing agent in the presence of asolvent and a catalyst to produce compound I-7:

(c) reacting compound I-7 with phosphate compound A-4:

to produce compound I-8:

wherein the reaction is carried out at a temperature of about 20 to 40°C. and in the presence of a solvent selected from the group consistingof THF, MTBE, CH₂Cl₂, MeCN, toluene and a mixture comprising two of theforegoing solvents;

(d) reducing compound I-8 in the presence of a catalyst selected fromthe group consisting of Pd, Raney Ni, Pt, PdCl₂, and Pd(OH)₂ to producecompound I-9:

(e) reacting compound I-9 with an acid to produce compound I-10:

and

(f) reducing compound I-10 in the presence of a catalyst to producecompound I-11:

wherein P¹ is Boc and P² is selected from the group consisting of Ac,Bn, Boc, Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts.

Compound I-11 can be used as an intermediate compound for makingcompounds of Formula (I) or Formula (II):

Also described herein is a process of making compound I-12:

comprising reacting compound I-14:

with compound I-15:

wherein R² and R³ are each independently selected from the groupconsisting of C₁₋₆alkyl, benzyl, and phenyl. In one embodiment, R² andR³ are each independently selected from the group consisting of methyl,ethyl, propyl, butyl and benzyl. In another embodiment, R² and R³ areboth methyl.

In one embodiment, the above process for making compound I-12 comprises2 steps:

(a) reacting compound I-14 with compound I-15 to produce compound i-17:

and

(b) hydrolyzing compound I-17 to produce compound I-12.

The above step (a) can be carried out in the presence of a solvent.Suitable solvents include, but are not limited to, ethyl benzene,toluene, trifluorotoluene, xylenes, cumene, and tert-butyl benzene. Inone embodiment, the solvent is ethyl benzene.

The above step (a) can be carried out at a temperature of 110° C. to150° C., or more specifically, 125° C. to 135° C. In one embodiment, thetemperature is 125° C. to 135° C.

The above hydrolysis step (b) can be carried out in the presence of abase. Suitable bases include, but are not limited to, NaOH, LiOH, KOH,CsOH, Ca(OH)₂, Ba(OH)₂, Mg(OH)₂, K₂CO₃, Na₂CO₃, and Cs₂CO₃. In oneembodiment, the base is NaOH.

The above step (b) can be carried out in the presence of a solvent.Suitable solvents include, but are not limited to, methanol, water, THF,EtOH, IPA, α-methyl-THF, and mixtures thereof. In one embodiment, thesolvent is the mixture of methanol/water, THF/water, EtOH/water,IPA/water, or α-methyl-THF/water. In another embodiment, the solvent isa mixture of methanol/water.

In one embodiment, compound I-14 can be prepared from reacting compoundI-13:

with (MeO)₂SO₂, wherein R² is as defined above.

In one embodiment, R² is selected from the group consisting of methyl,ethyl, propyl, butyl and phenyl. In another embodiment, R² is methyl.

In one embodiment, the above step from compound I-13 to compound I-14 iscarried out without a solvent.

In another embodiment, the above step from compound I-13 to compoundI-14 is carried out at a temperature of 10° C. to 85° C., or morespecifically, 25° C. to 65° C. In one embodiment, the temperature is 25°C. to 65° C.

Further described herein is a process of making a compound of Formula(I):

comprising reacting compound I-11 with compound I-12.

The reaction between I-11 and I-12 can be carried out in the presence ofa coupling reagent. Suitable coupling reagents include, but are notlimited to, CDI, DCC, EDC, EDC methiodide, T3P, HATU, HBTU andmix-anhydrides. In one embodiment, the coupling reagent is EDC.

The reaction between I-11 and I-12 can be carried out in the presence ofa solvent while the substrate is treated with an acid such as HCl,MeSO₃H, H₂SO₄ to selectively protect the secondary pyrrolidine amine.Suitable solvents include, but are not limited to, both aqueous andnon-aqueous solvents such as MeOH, EtOH, IPA, n-PrOH, MeCN, DMF, DMAc,THF, EtOAc, IPAc, or toluene.

A promoter can be used in the reaction between I-11 and I-12. Suitablepromotors include, but are not limited to, HOBT and HOPO.

Suitable pH values for the reaction between I-11 and I-12 can be 2.5 to5.0, or more specifically, 3.0 to 4.0, or even more specifically, 3.0 to3.5. The pH can be adjusted to the desired ranges using an acid such asHCl, HBr, HI, HNO₃, H₂SO₄, H₃PO₄, TFA and MeSO₃H. In one embodiment, thepH is 3.0 to 3.5. In another embodiment, the pH is 3.3 to 3.5.

Also described herein is a process of making a compound of Formula (II):

comprising reacting compound I-11 with a suitable salt of compound I-30:

In one embodiment, the salt of compound I-30 is the lithium salt.

In one embodiment, the reaction between I-11 and I-30 is carried out inthe presence of an acid. In one embodiment, the solvent is selected fromthe group consisting of HCl, HBr, HI, HNO₃, H₂SO₄, H₃PO₄, TFA andMeSO₃H.

In one embodiment, the reaction between I-11 and I-30 is carried out inthe presence of a solvent. In one embodiment, the solvent is selectedfrom the group consisting of MeOH, EtOH, IPA, n-PrOH, MeCN, DMF, DMAc,THF, EtOAc, IPAc, or toluene.

The lithium salt of compound I-30 can be prepared from ethyl pyruvate(compound i-37) through multiple step reactions as illustrated in Scheme4 and Example 4:

As used herein, the term “alkyl” means both branched- and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms. For example, C₁₋₆alkyl includes, but is not limited to,methyl (Me), ethyl (Et), n-propyl (Pr), n-butyl (Bu), n-pentyl, n-hexyl,and the isomers thereof such as isopropyl (i-Pr), isobutyl (i-Bu),secbutyl (s-Bu), tert-butyl (t-Bu), isopentyl, sec-pentyl, tert-pentyland isohexyl.

As used herein, the term “aryl” refers to an aromatic carbocycle. Forexample, aryl includes, but is not limited to, phenyl and naphthale.

Throughout the application, the following terms have the indicatedmeanings unless noted otherwise:

Term Meaning Ac Acyl (CH₃C(O)—) Aq Aqueous Bn Benzyl BOC (Boc)t-Butyloxycarbonyl Boc₂O Di-tert-butyl dicarbonate Bz Benzoyl ° C.Degree celsius Calc. or Calculated calc'd Cbz Carbobenzyloxy CDI1,1′Carbonyldiimidazole DCC N,N′-Dicyclohexycarbodiimide DCMDichloromethane DKR Dynamic kinetic resolution DMAcN,N-dimethylacetamide DMAP 4-Dimethylaminopyridine DMFN,N-dimethylformamide DMPM 3,4-Dimethoxybenzyl DMSO Dimethyl sulfoxideEDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide Eq. or Equivalent(s)equiv. ES-MS and Electron spray ion-mass spectroscopy ESI-MS Et EthylEtOAc Ethyl acetate FMOC 9-Fluorenylmethyloxycarbonyl g Gram(s) h or hrHour(s) HATU O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate HBTUO-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate)HCl Hydrogen chloride HMDS Hexamethyldisilazane HPLC High performanceliquid chromatography HOAc Acetic acid HOBT 1-Hydroxy-1H-benzotriazoleHOPO 2-Hydroxypyridine-N-oxide IPA Isopropyl alcohol kg Kilogram(s)LC/MS or Liquid chromatography mass spectrum LC-MASS L Liter(s) LAH orLithium aluminium hydride LiAlH₄ LCAP Liquid Chromatography Area PercentLiBH₄ Lithium borohydride M Molar(s) Me Methyl MeCN Acetonitrile MeOHMethanol min Minute(s) mg Milligram(s) mL Milliliter(s) mmolMillimole(s) Moz or p-Methoxybenzyl carbonyl MeOZ MTBE Methyl tert-butylether NADP Nicotinamide adenine dinucleotide phosphate sodium salt nMNanomolar Ns 4-Nitrobenzene sulfonyl PCC Pyridinium chlorochromate 5%Pd/C Palladium, 5 weight percent on activated carbon Ph Phenyl r.t. orrt RT or RT Sat. Saturated TBDMSCl Tert-Butyldimethylsilyl chloride TEAor Triethylamine Et₃N TEMPO 1-Oxyl-2,2,6,6-tetramethylpiperidine TESClTriethylchlorosilane TFA Trifluoroacetic acid THF Tetrahydrofuran TMSClTrimethylchlorosilane Ts p-Toluene sulfonyl

Reaction Schemes below illustrate the synthetic steps, reagents andconditions employed in the synthesis of the compounds described herein.The synthesis of the compounds of Formula (I), (II), I-11, I-12 and I-30which are the subject of this invention may be accomplished by one ormore of similar routes.

Example 1 Preparation of Compound i-11 from Starting Compound I-5b

In Scheme 1, starting material compound I-5b was converted to compoundi-6 by reacting with acetone and Boc₂O.

Once compound i-6 was obtained, it was converted to i-7 by TEMPOoxidation. For the TEMPO oxidation and subsequent HWE coupling step, aone-pot through process was used such that the crude steam of thealdehyde i-7 after phase cut was used directly for the HWE reaction toavoid solvent switch. Unsaturated ketone i-8 was isolated over 5 steps.Finally, compound i-8 was converted to compound i-11 through i-9 andi-10. Detailed experimental conditions are described below.

Step 1. Preparation of Acetonide-Boc Alcohol i-6 from I-5b

To a flask equipped a Dean-Stark trap was charged(1R,2R)-(−)-2-amino-1-phenyl-1,3-propanediol (I-5b) (10 g, 58.6 mmol),acetone (12.0 ml), and toluene (40.0 ml) (or MTBE). The slurry washeated to reflux for 22 h. To the solution was added di-tert-butyldicarbonate (14.2 g, 64.5 mmol) at RT. The mixture was stirred at RT for3.5 h, and to the mixture was added 1.5 g Boc₂O, then continued stirringovernight. The mixture was concentrated to 23.5 g oil, flushed with 40mL heptane and concentrated to 23.7 g oil.

To the resulting mixture was added 18 mL heptane and the solution wasseeded with 35 mg compound i-6. Crystalline seed bed initiated within 10min. The resulting mixture was placed in −20° C. freezer overnight andthen filtered and washed with 20 mL −20° C. heptane.

The wet cake was vacuum dried at 22° C. under N₂ overnight to afford14.13 g compound i-6 as a beige solid (78.5%). Melting point (MP) was69-72° C.

¹H NMR (CDCl₃) δ 7.45-7.30 (m, 5H), 4.80 (br s, 1H), 4.58 (br s, 1H),3.82 (br m, 2H), 3.51 (br m, 1H), 1.70 (s, 3H), 1.60 (s, 3H), 1.52 (s,9H); ¹³C NMR (CDCl₃) δ 154.5, 136.9, 129.2, 128.9, 127.6, 94.8, 81.7,78.4, 67.9, 63.8, 28.5, 27.8, 26.1. Anal. Calcd. for C₁₇H₂₅NO₄: C,66.43; H, 8.20; N, 4.56. Found: C, 66.33; H, 8.43; N, 4.59.

HPLC Method

Column: Waters Xbridge C18, 50 mm×4.6 mm, 2.5 μm particle size;

Column Temp.: 25° C.; Flow rate: 1.0 mL/min; Detection: 210 nm & 254 nm;

Mobile phase: A: 1.0 mL of NH₄OH (28% as NH₃) dissolved in 1 L of water;B: MeCN

Mobile Phase Program:

Time, min 0 4 8 12 16 16.5 20 A % 100 60 60 50 5 100 100 B % 0 40 40 5095 0 0

Step 2. Preparation of Compound i-7 by TEMPO Oxidation of Compound i-6

To a solution of i-6 alcohol in toluene (20 g assay, ˜60 mL) was addedacetonitrile (120 mL) at RT. KBr (1.16 g), NaHCO₃ (1.8 g) and water (40mL) were then charged resulting in a biphasic mixture. The biphasicmixture was cooled to 5° C. and TEMPO (305 mg) was added. Then, NaClOsolution (Clorox; 6 wt %; 101 g) was added dropwise at 0-5° C. over 2 h.After addition, the reaction was stirred at 5° C. for ˜30 min.Conversion of >96% was obtained.

The reaction was quenched by dropwise addition of 10% sodium sulfite (50mL) at 5° C. The organic layer was separated and directly used for thesubsequent HWE coupling step without further purification. The assayyield was 17.5 g (88%) by ¹H NMR using DMAc as internal standard.

Retention times of i-6 and i-7 using the following HPLC method wereabout 3.3 min and 3.9 min, respectively.

HPLC Method

Column: Zorbax, Eclipse Plus C18, 4.6×50 mm, 1.8 μm particle size;

Column Temperature: 22° C.; Flow Rate: 1.5 mL/min; UV Detection: 210 nm;

Mobile Phase: A: 95/5/0.1, H₂O/Methanol/H₃PO₄ B: 95/5, MeCN/methanol

Mobile Phase Program:

Time, min 0 5 6 A % 60 10 10 B % 40 90 90

Step 3. Preparation of i-8 by Horner Wadsworth Emmons (HWE) CouplingReaction

To a solution of i-7 aldehyde in wet toluene/acetonitrile (162 gsolution; 17.5 g assay; 10.81 wt %) obtained above at −10° C. were addedacetonitrile (140 mL), phosphonate a-4 (24.6 g) and LiBr (14.9 g) whilethe internal temperature was maintained below 0° C.

The reaction was warmed up to 0° C., and Hunig's base (22.2 g) wascharged at 0-5° C. dropwise over 2 h. The resulting reaction mixture wasstirred at 0-5° C. for 2-4 h and allowed to warm to RT, followed byaging at RT for 12 h. HPLC showed conversion(product/(product+aldehyde)) of >99%.

The slurry was cooled to 5° C., and a 10% aqueous solution of citricacid (˜75 g) was added dropwise to adjust the pH to 6.5-7.0 whilemaintaining the batch temperature at 0-5° C. The aqueous phase wasseparated at 0-5° C. and discarded.

The organic layer was washed with saturated NaHCO₃ (57 mL) and with H₂O(57 mL) successively. The organic phase was solvent switched to IPA to afinal volume of ˜192 mL. The product was gradually crystallized duringthe distillation.

Water (16.4 mL, 0.6 vol.) was added, and the resulting slurry was heatedto 49° C. to give a homogeneous solution. The resulting solution wascooled to 40° C. and seeded (0.27 g). The resulting mixture was aged at40° C. for 2 h to establish a seed bed, and H₂O (93 mL) was chargeddropwise at 40° C. over 3 h, followed by aging at 40° C. for 1 h. Theslurry was allowed to cool to 5-10° C. over 2 h, followed by aging at5-10° C. for 2 h.

The wet cake was washed with 50% H₂O/IPA (a 164 mL cold displacementwash followed by a 110 mL slurry wash). Suction dried under nitrogengave the product as an off-white solid (24.9 g, 100 wt %, >99 LCAP, 80%isolated yield from aldehyde).

Using the following HPLC method, the retention times of i-7, a-4 and i-8were about 3.0 min, 1.2 min and 3.8 min, respectively.

HPLC Method

Column: Zorbax, Eclipse Plus C18, 4.6×50 mm, 1.8 μm particle size

Column Temp: 40° C.; Flow Rate: 1.5 mL/min; UV Detection: 210 nm;

Mobile Phase: A: 0.1% H₃PO₄ B: MeCN

Mobile Phase Program:

Time, min 0 3 7 A % 60 10 10 B % 40 90 90

Step 4. Preparation of Compound i-9 from Compound i-8

THF (84 g) followed by enone i-8 (19.07 g) and 10% Palladium on carbon(0.95 g) were charged to a hydrogenation vessel. The batch washydrogenated for 90 min at 25° C. until uptake of hydrogen had ceased.The catalyst was removed through filtration of a bed of solka floc. Thefiltered residues were washed with THF (84 g). The combined organicphase was solvent switched to IPA to a final volume of 142 mL, which wasdirectly used in the next step. Assay yield of 93% was obtained (17.8 gof i-9).

Using the following HPLC method, the retention times of i-8 and i-9 wereabout 11.2 min and 11.4 min, respectively.

HPLC Method

Column: HiChrom ACE C18 (250×4.6 mm), 3 μm particle size;

Column Temperature: 30° C.; Flow rate: 1.0 mL/min; Detection: 210 nm,254 nm;

Mobile phase: A: 1 mL of phosphoric acid (85%) dissolved in 1 L of H₂OB: MeCN

Mobile Phase Program:

Time, min 0 5 8 15 16 20 A % 95 65 5 5 95 95 B % 5 35 95 95 5 5

Step 5. Preparation of Compound i-10 from Compound i-9

To a solution of the N-Boc-Ketone aniline i-9 (26.1 assay kg) in IPA(˜125 g/L) was added 4N HCl in IPA (220.8 L) at RT. The reaction mixturewas stirred vigorously at 20-25° C. for 24 h. The batch was distilledunder reduced pressure, at constant volume by charging IPA up to onebatch volume, to remove HCl. The batch was then concentrated to a finalvolume of ˜215 L.

The resulting slurry was heated to 45° C., and IPAc (˜430 L) was slowlyadded to the batch over 2-3 h. The slurry was then cooled to ˜20° C.over 1-2 h and aged overnight. The batch was filtered, and the cake waswashed with a 1:2 mixture of IPA:IPAc (52 L) followed by IPAc (52 L).The wet cake was dried at 45° C. under nitrogen atmosphere to give thecyclic imine bis-HCl monohydrate salt i-10 (16.1 kg). The isolated yieldof 94% was obtained.

Using the same HPLC method as in Step 7 (i-8 to i-9), the retentiontimes of i-9 and i-10 (bis-HCl salt) were about 11.3 min and 8.3 min,respectively.

Step 6. Preparation of Compound i-11 from Compound i-10

To a mixture of imine dihydrochloride monohydrate i-10 (12.0 g, 98.5 wt%) in THF (86 mL) under N₂ was added hexamethyldisilazane (10.95 g)while maintaining the batch temperature below 25° C. The resultingslurry was stirred vigorously at ambient temperature for 2 h.

A 300 mL autoclave was charged with a suspension of 5% platinum onalumina (0.605 g) in THF (32 mL), followed by the substrate slurryprepared above. The resulting mixture was stirred at RT under hydrogen(40 psig) until the hydrogen uptake ceased. The completion of thehydrogenation was confirmed by HPLC, and the vessel was inerted withnitrogen.

The reaction mixture was discharged, and the vessel rinsed with 96 mL ofTHF. The batch was filtered through a pad of Solka Floc, and the pad wasrinsed with the THF vessel rinse (˜96 mL). The combined filtrate wasstirred with 0.5 M hydrochloric acid (129 mL) at ambient temperature for1 h. The aqueous layer was separated. IPAc (39 mL) followed by 5 Nsodium hydroxide (˜15 mL) was added to adjust the pH to 10.0 withvigorous stirring.

The organic layer (˜120 mL) was separated and treated with AquaGuardPowder (Meadwestvaco) (2.4 g) at RT for 2 h. The solution was filteredthrough a pad of Solka Floc, and the pad was rinsed with 2-propanol (18mL). The combined filtrate was concentrated to 70 mL. The solution wasdistilled at the constant volume by feeding a total of 140 mL of2-propanol, maintaining the batch temperature at 33-35° C. The resultingsolution was concentrated to ˜34 mL and heated to 50° C., followed byaddition of H₂O (6.3 mL). The resulting solution was cooled to 41-43° C.and seeded with pyrrolidine aniline hemihydrate (42 mg). The resultingmixture was aged at 41-43° C. for 1 h to establish a seed bed.

Water (60.9 mL) was charged at 41-43° C. over 6 h, and the resultingmixture was allowed to cool to 10° C. over 3 h, followed by aging at 10°C. for 2 h. The solids were collected by filtration and washed with 25%2-propanol/H₂O (50 mL). The wet cake was suction-dried at ambienttemperature under nitrogen to afford 7.68 g of pyrrolidine aniline i-11as hemihydrate.

¹H NMR (d₆-DMSO) δ 7.27 (m, 4H), 7.17 (m, 1H), 6.81 (d, J=8.1, 2H), 6.45(d, J=8.1 Hz, 2H), 5.07 (s, br, 1H), 4.75 (s, 2H), 4.18 (d, J=7.0 Hz,1H), 3.05 (m, 2H), 2.47 (dd, J=13.0, 6.7 Hz, 1H), 2.40 (dd, J=13.0, 6.6Hz, 1H), 1.53 (m, 1H), 1.34 (m, 1H0, 1.22 (m, 2H).

¹³C NMR (d₆-DMSO) δ 146.5, 144.3, 129.2, 127.8, 127.4, 126.8, 126.7,114.0, 76.8, 64.4, 60.1, 42.1, 30.2, 27.2.

Using the following HPLC method, the retention times of i-10 (bis-HClsalt) and i-11 were about 8.3 min and 8.5 min, respectively.

HPLC Method

Column: Waters Xbridge C18, 150×4.6 mm, 3.5 μm;

Column Temperature: 25° C.; Flow rate: 1 mL/min; Detection: 210 nm, 254nm;

Mobile phase: A: Acetonitrile B: 0.1% aqueous NH₄OH adjusted to pH9.5with H

Mobile Phase Program:

Time, min 0 4 8 10 17 A % 99 65 65 30 30 B % 1 35 35 70 70

The crystalline anhydrous and hemihydrate forms of the pyrrolidineaniline compound i-11 were characterized by powder x-ray diffraction(PXPD) and shown in FIG. 1 and FIG. 2, respectively.

The crystalline anhydrous form of the pyrrolidine aniline compound i-11was characterized by XRPD by the following reflections with thed-spacing and corresponding intensities listed below.

Position [°2 Theta] d-spacing [Å] Relative Intensity [%] 17.8453 4.97100 25.1979 3.53 51.24 20.1002 4.42 39.04 23.9931 3.71 32.65 16.70735.31 27.98 25.5483 3.49 20.21 19.6576 4.52 20.2 13.8883 6.38 20.0828.086 3.18 18.72 20.6498 4.30 16.23

The crystalline hemihydrate of the pyrrolidine aniline compound i-11 wascharacterized by XRPD by the following reflections with the d-spacingand corresponding intensities listed below.

Position [°2 Theta] d-spacing [Å] Relative Intensity [%] 17.9681 4.94100 17.8666 4.96 80.62 23.1905 3.84 73.82 15.3049 5.79 71.53 19.79554.49 65.46 19.9483 4.45 56 23.1076 3.85 54.38 25.3415 3.51 53.04 16.08595.51 44.07 25.6746 3.47 41.85

Example 2 Process for Making Compound i-12 from Compound i-14 andCompound i-15

Step 1. Preparation of 3-Aza-tricyclo[4.2.1.0^(2,5)]non-7-en-4-one(beta-Lactam) i-15 from i-18

In a 100 L RBF fitted with an overhead stirrer, a thermocouple and anitrogen inlet, was charged 36.8 L of DCM and 8.83 L of norbornadienei-18. The solution was cooled to −15° C. A solution of 7.92 L ofchlorosulfonylisocyanate in 11.2 L of DCM was added at a rate that keepsthe internal temperature <5° C. The mixture was warmed to RT. Afterreaction was completed (by NMR), the reaction mixture was quenched intoa 170 L cylinder vessel containing sodium sulfite (10.7 kg) in water(35.7 L) solution at a rate that keeps the internal temperature <15° C.,maintaining a pH between 8.5 to 9.0 by addition of NaOH. Final pH wasadjusted at 8.5.

Acetonitrile (24 L) was added and the layers were separated. If needed,24 L of 20% brine solution was added to facilitate the viscous aqueouslayer to flow. The top organic layer was separated and concentrated to24 L and then filtered through an in-line filter into a 50 L RBF. At theprep area, removing residual inorganic salts via in-line filtration wasproblematic due to premature crystallization of the product. Moreacetonitrile and decanting at higher temperature were found helpful.

The solution was concentrated to 16 L and solvent switched to heptane.The precipitate was filtered and washed with 1 vol heptane. The solidwas dried overnight in a vacuum oven at 45° C. Isolated 8.8 kg of theproduct (77% isolated yield as 100 wt %)

Alternative Work-Up Procedure

In a 1 L 3-neck RBF fitted with an addition funnel, a thermocouple, amagnetic stirrer, and a nitrogen inlet, was charged 184 mL DCM and 44.2mL norbornadiene i-18. The solution was cooled to −12° C. A solution of39.6 mL chlorosulfonylisocyanate in 56 mL DCM was added via the additionfunnel at a rate that maintained a temperature range of −10 to 1° C.After the addition the mixture was allowed to warm to RT over 1-2 h. Thereaction was monitored by ¹H NMR showing the disappearance of thenorbornadiene.

Work Up

In a 2 L 4-neck RBF fitted with a mechanical stirrer, an addition funneland a pH probe, was charged 53.6 g sodium sulfite and 680 mL (17 vol)water. The reaction mixture was added via the addition funnel whilesimultaneously adding 10N NaOH keeping the temperature range −2 to 14°C. and pH>8.0. After the addition was complete the pH was adjusted to pH8.5 and the mixture was allowed to warm to 15° C.

To the mixture was added 240 mL sec-BuOH. Organic layer was separated.The aqueous was back extracted 1× with 200 mL sec-BuOH.

Crystallization

In a 500 mL 3-neck RBF fitted with a distillation head temp probe and amechanical stirrer, the combined organic solution was concentrated to200 mL (5 vol) under vacuum with solution temperature kept at 25-27° C.(bath temp at 80° C.) bp=23° C. The solution was solvent switched totoluene till the ratio of toluene:BuOH=97:3 and the KF<200 ppm.

The slurry was cooled to 27° C. and to which was added 120 mL (3 vol)heptane dropwise via an addition funnel and aged overnight at roomtemperature.

The resulting mixture containing compound i-15 was filtered and washedwith 1× w/40 mL (1 vol) heptane and dried in a vacuum oven at 40° C.overnight to yield compound i-15.

Step 2. Preparation of Compound i-14 from i-13

To a 20 L cylinder reactor equipped with an overhead stirrer,thermocouple, and nitrogen inlet was charged(S)-(+)-2-pyrrolidone-5-carboxylate i-13 (6.04 kg, 97 wt %), anddimethyl sulfate (5.33 L). The resulting reaction mixture was stirred at53-58° C. for 12-15 h to afford product i-14 (>90 LCAP % conversion).The reaction mixture was cooled 25-30° C.

HPLC Method

Column: Zorbax Eclipse Plus C18 50×4.6 mm, 1.8 μm particle size;

Column Temp.: 25° C.; Flow Rate: 1.5 mL/min; Detection: 230 nm;

Mobile Phase: A: Water 0.1% H₃PO₄ B: Acetonitrile

Mobile Phase Program:

Time, min 0 5 6 A % 90 5 5 B % 10 95 95

To a 50 L room bottom reactor, equipped with an overhead stirrer,thermocouple, and nitrogen inlet, was charged triethylamine (8.93 L),and cooled to 10-15° C. The above reaction mixture was slowly added toTEA at 15-25° C. over 1 h, and stirred at RT for 0.5 h. The reactionmixture was transferred to a 100 L extractor, which contained toluene(40 L) and water (10 L).

After phase separation, the aqueous layer was extracted with toluene(1×20 L). The combined organic layers were washed with 10% NaHCO₃ (2×5L) and brine (5 L). The organic layer was azotropically concentrated toafford an oil crude product methyl(2S)-5-methoxy-3,4-dihydro-2H-pyrrole-2-carboxylate (i-14) in toluenesolution (expect KF<300 ppm, kg, 6.60 kg, 72.3 wt %, 74% yield aftercorrection), which will be used in the next step.

Step 3. Preparation of Compound i-17 Through Cycloaddition/RetroDiels-Alder

To a 50 L cylinder reactor, equipped with an overhead stirrer,thermocouple, nitrogen inlet, and Dean-Stark, was charged methyl(2S)-5-methoxy-3,4-dihydro-2H-pyrrole-2-carboxylate i-14 (6.60 kg, 72.3wt %), beta-lactam i-15 (4.19 kg), and ethyl benzene (9.54 L). Theresulting reaction mixture was stirred at 128° C. for 48 h. During thereaction, some low boiling point by-product such as methanol was removedthrough Dean-Stark in order to reach the interior temperature at 128° C.In prep lab, the internal temperature was 119-120° C., and the reactionwas stirred at this temperature for 80 h (92% conversion by ¹H NMR).

The reaction mixture was cooled to 35° C., diluted with toluene (14.3 L,3 V) and Darco G-60 (1.43 kg) was added. The resulting mixture wasstirred at the same temperature for 1 h. The Dacro G-60 was filtered offby passing through solka flock, washed with toluene (19.1 L). Assayproduct i-17 in the toluene solution was 3.81 kg (65%).

The combined filtrates were concentrated and purified by silica gelpluge (22.5 kg silica gel, eluted by heptane 5 V; acetone/heptane=1:2,15 V; acetone/heptane=2:1, 18 V).

The resulting product-rich solution was concentrated, andsolvent-switched to EtOAc (6.5 L, total volume). Crystalline producti-17 was formed during solvent-switch to EtOAc. MTBE (7 L) was addedslowly over 1 h (at this point, the ratio of EtOAc:MTBE was about 1:4 by¹H NMR). The resulting slurry was stirred at 5-10° C. for 1 h. Thecrystalline product i-17 was collected by filtration, washed with coldMTBE/EtOAc (5:1, 1 L), MTBE (3 L), dried under vacuum with nitrogensweep to afford product i-17 (2.57 kg, >99 A % purity, 68% recoveredyield, or 44% isolated yield from i-14). MP was 88 to 89° C.

The crystalline i-17 was important for the ee % upgrade, crystallizationand isolation of product i-12 in the next step. Otherwise, the finalstep may require chiral separation or enzyme resolution.

HPLC Method

Column: Zorbax Eclipse Plus C18 50×4.6 mm, 1.8 μm particle size;

Column Temp.: 30° C.; Flow Rate: 1.5 mL/min; Detection: 230 nm;

Mobile Phases: A: Water 0.1% H₃PO₄ B: Acetonitrile

Mobile Phase Program:

Time, min 0 5 6 A % 90 5 5 B % 10 95 95

Step 4. Preparation of Compound i-12 Through Hydrolysis of Compound i-17

To a 50 L round-bottom, equipped with an overhead stirrer, thermocouple,nitrogen inlet, was charged methyl ester i-17 (4.70 kg), methanol (14.1L), and water (9.4 L). The resulting homogenous solution was cooled to0° C. 3 N sodium hydroxide (8.41 L) was slowly added through a pump atthe rate 28 mL/min while maintaining the internal temperature at 0° C.to 5° C. After complete addition of the sodium hydroxide, the reactionmixture was stirred at 0° C. to 5° C. until the reaction was completed.The reaction mixture was adjusted to pH=6.5-7.0 with 5 N HCl.

The reaction mixture was concentrated and azotroped with toluene to athick solution, and then solvent-switched to IPA. And the IPA solutionwas continued to azotrope to KF≦6 wt % and adjusted to a total volume(14.1 L) with IPA. The resulting slurry was stirred at 0° C. to 5° C.for 1-2 h. A crystalline product i-12 as hydrate (3 equiv of water) wascollected by filtration, washed with cold IPA (6 L), toluene (6 L), anddried under vacuum with nitrogen sweep overnight.

At this point, the crystalline hydrate product i-12 was continuallydried in an oven at 50 0° C. to 55° C. under vacuum with flowingnitrogen for 50 h.

The crystalline compound of i-12 easily absorbs moisture in the air toform a hydrate. MP of the hydrate is 69.5 0° C. to 70.5° C.

HPLC Method

Column: Waters, AtlantisHPLC Silica 150×4.6 mm column, 3 μm particlesize,

Column Temp.: 40° C. Flow rate: 1.00 mL/min; Detection: 210 nm;

Mobile Phase: A: Water 0.1% H₃PO₄ B: Acetonitrile

Mobile Phase Program:

Time, min 0 5 6 A % 90 5 5 B % 10 95 95

Example 3 Preparation of Compound of Formula (I) from Compound i-11 andCompound i-12

To a three neck flask equipped with a N₂ inlet, a thermo couple probewas charged pyrrolidine i-11 (10.0 g), sodium salt i-12 (7.87 g),followed by IPA (40 mL) and water (24 mL). 5 N HCl (14.9 mL) was thenslowly added over a period of 20 min to adjust pH=3.3-3.5, maintainingthe batch temperature below 35° C. Solid EDC hydrochloride (7.47 g) wascharged in portions over 30 min. The reaction mixture was aged at RT foradditional 0.5-1 h, aqueous ammonia (14%) was added dropwise to pH˜8.6.The batch was seeded and aged for additional 1 h to form a slurry bed.The rest aqueous ammonia (14%, 53.2 ml total) was added dropwise over 6h. The resulting thick slurry was aged 2-3 h before filtration. Thewet-cake was displacement washed with 30% IPA (30 mL), followed by 15%IPA (2×20 mL) and water (2×20 mL). The cake was suction dried under N₂overnight to afford 14.3 g of compound of Formula (I).

¹H NMR (DMSO) δ 10.40 (s, NH), 7.92 (d, J=6.8, 1H), 7.50 (m, 2H), 7.32(m, 2H), 7.29 (m, 2H), 7.21 (m, 1H), 7.16 (m, 2H), 6.24 (d, J=6.8, 1H),5.13 (dd, J=9.6, 3.1, 1H), 5.08 (br s, OH), 4.22 (d, J=7.2, 1H), 3.19(p, J=7.0, 1H), 3.16-3.01 (m, 3H), 2.65 (m, 1H), 2.59-2.49 (m, 2H), 2.45(br s, NH), 2.16 (ddt, J=13.0, 9.6, 3.1, 1H), 1.58 (m, 1H), 1.39 (m,1H), 1.31-1.24 (m, 2H).

¹³C NMR (DMSO) δ 167.52, 165.85, 159.83, 154.56, 144.19, 136.48, 135.66,129.16, 127.71, 126.78, 126.62, 119.07, 112.00, 76.71, 64.34, 61.05,59.60, 42.22, 31.26, 30.12, 27.09, 23.82.

HPLC Method—for Monitoring Conversion

Column: XBridge C18 cm 15 cm×4.6 mm, 3.5 μm particle size;

Column Temp.: 35° C.; Flow rate: 1.5 mL/min; Detection: 220 nm;

Mobile phase: A. 5 mM Na₂B4O₇.10 H20 B: Acetonitrile

Gradient:

Time, min 0 6 8 10 A % 30 30 5 5 B % 70 70 95 95HPLC Method—for Level of Amide Epimer DetectionColumn: Chiralpak AD-H 5 μm, 250 mm×4.6 mm.Column Temp: 35° C.; Flow rate: 1.0 mL/min; Detection: 250 nm;Mobile phase: Isocratic 30% Ethanol in hexanes+0.1% isobutylamine

Example 4 Preparation of Compound i-30 from Compound i-37

Step 1. From Compound i-37 to Compound i-36

To a solution of tert-butyl carbazate (109.37 g, 1.0 mol eq) and aceticacid (54.7 g, 1.1 mol eq) in MTBE (656 mL) at 0° C. was added ethylpyruvate (96.0 g, 1.0 mol eq) over 2 h. The resulting slurry was aged at0-5° C. for 3 h. The reaction was exothermic. The product began tocrystallize out during the addition of ethyl pyruvate.

The resulting solids were collected by filtration, and the wet cake waswashed with cold MTBE (220 mL displacement wash and 440 mL slurry wash)and suction-dried under N₂ to afford 172 g of the Boc-hydrazone compoundi-36 as white solids. 90% Isolated yield. 8.6 g liquor losses (5%).

The concentration of i-36 in the supernatant prior to filtration was 18mg/mL. The retention time of Boc-hydrazone using the following HPLCmethod was about 11.7 min.

HPLC Method—Achiral Method

Column: Phenomenex Luna C8 (250×4.6 mm I.D., 5 μm);

Detector: UV 205 nm; Oven: 40° C.; Flow rate: 1.0 mL/min; Injection vol:10 μL;

Mobile phase A: 0.1% H₃PO₄ in Water (v/v); B: ACN

Gradient program:

Time, min 0 8 15 20 A % 95 40 5 5 B % 5 60 95 95

Step 2. From Compound i-36 to Compound i-35

In a nitrogen-filled glovebox, Rh(nbd)₂BF₄ (374 mg, 1.00 mmol, 1.0 mol%) and SL-W008-1 (990 mg, 1.05 mol %) were weighed into a glass vial.Then 22 mL of degassed EtOH were added to give a homogeneous solutionwhich was aged 16 h at 22° C. A slurry of 23.0 g (100 mmol)Boc-hydrazone i-36 in 100 mL of EtOH was prepared. This slurry was thencharged to a 300 mL autoclave with a 20 mL EtOH flush. Degassed withvacuum/nitrogen purges, then charged the catalyst solution undernitrogen with a 10 mL EtOH flush. Hydrogenated at 500 prig H₂ for 48 hat 20-25° C. HPLC assay reveals 85% assay yield.

The batch was kept under nitrogen even after the reaction was complete.The product underwent oxidation to give the Boc-hydrazine in thepresence of oxygen and rhodium. The target HPLC conversion is 96%(product/(product+starting material), at 210 nm), which corresponds to99.3 mol % conversion.

Using the Achiral HPLC method described in Step 1, the retention timesof i-35 and i-36 were about 11.5 min and 11.7 min, respectively.

Using the following Chiral HPLC method, the retention times of i-36,i-35 and the undesired hydrazide product were about 2.9 min, 3.8 min and4.2 min, respectively.

Chiral Method

Chiralpak AD-RH, 2.5 mm×15 cm;

Mobile Phase: A=MeCN; B=0.1% (v/v) H3PO4 (aq)

1.0 mL/min; 1.0 uL injection, 35 C, 210 nm, 14 min runtime, 0.2 min posttime

Gradient:

Time, min 0 1 7 8 10 10.2 14 A % 40 40 60 80 80 40 40 B % 60 60 40 20 2060 60

Step 3. From Compound i-35 to Compound i-34

A solution of Boc-hydrazine i-35 (23.6 g assay, 1 mol eq) in EtOH (190mL) was degassed by repeating an evacuation/N₂ refill cycle and treatedwith methanesulfonic acid (14.67 g, 1.5 mol eq) at 60° C. for 15 h untilthe consumption of the starting material (Boc-hydrazine) was confirmedby ¹H NMR. The resulting solution was concentrated to give the MSA saltof the deprotected hydrazine i-34 as an oil (34.51 g). The product wassubjected to the subsequent cyclization step without furtherpurification.

The targeted mol % conversion is 99% by ¹H NMR. The presence of oxygencan cause degradation of substrate/product. The reagents charges in thesubsequent cyclization step were calculated by assuming 100% yield forthis de-Boc step.

Step 4. From Compound i-34 to Compound i-32

A crude solution of the deprotected hydrazine i-34 (10.0 g as the freebase) in EtOH was concentrated to ˜38 mL (3.8 mL/g free base). Thesolution was distilled at the constant volume to remove EtOH whilefeeding toluene to give a biphasic solution.

The bottom layer contained the hydrazine MSA salt. The EtOH in thebottom layer was 0.7 mol eq (relative to the hydrazine) by ¹H NMR.

The resulting biphasic solution was diluted with CH₂Cl₂ (100 mL) andcooled to −45° C., followed by addition of ethyl acetamidate HCl (10.29g, 1.1 mol eq). N,N-diisopropylethylamine (27.39 g, 2.8 mol eq) wasadded dropwise over 1 h while maintaining the batch temperature between−45° C. and −40° C. The resulting suspension was allowed to warm to RTover 30 min and aged at RT for 2 h. The batch was cooled to −10° C., andtriethyl orthoformate (51.2 g, 10 mol eq) and formic acid (4.14 g, 1.5mol eq) were added dropwise while maintaining the batch temperaturebelow 0° C. The resulting mixture was distilled at 20-25° C. to collect100 mL of solvents. Formic acid (4.14 g, 1.5 mol eq) was chargeddropwise at RT, and the resulting mixture was heated to 70° C. for 4 huntil the HPLC conversion reached 96 A % (i-32/(i-32+i-33)).

Formic acid with good quality (98%) was used. The enantiopurity of theproduct was eroded from 95% ee to 93% ee. Ee will be eroded further byprelonged aging. The racemization gets faster at higher temperatures.Reactions at lower temperature were sluggish and gave lower conversion.

The reaction was allowed to cool to 10° C. and diluted with H₂O (25 mL),followed by aging at RT for 30 min to quench orthoformate. The pH of themixture was adjusted to 8 with 15% Na₂CO₃ aq (˜59.9 mL, 1.3 mol eq). Theresulting mixture was extracted with EtOAc (70 mL×3). The combinedorganic layer was washed with 25% NaCl aq (70 mL) and 1 M phosphatebuffer (pH 7, 70 mL). The solution was concentrated to ˜104 mL, and thesolvent was switched to 2-MeTHF by distillation while feeding a total of440 mL 2-MeTHF. The resulting hazy solution was filtered to removetriethylamine HCl salt (˜0.4 g). HPLC assay reveals 10.40 g of product(75% assay yield).

Quenching orthoformate was mildly exothermic and external cooling wasrequired to maintain the batch temperature below 25° C. The spec fortoluene level after solvent switch was 1.0 v/v %. Product losses inaqueous layers were typically <0.5% in the aqueous layer post backextractions and 2% in each brine and buffer wash. The buffer wash washelpful to promote the subsequent enzymatic resolution reaction.

Using the Achiral HPLC method described in Step 1, the retention timesof i-33, i-32 and the ethyl formate by-product were about 4.0 min, 8.3min and 8.4 min, respectively.

Step 5. From Compound i-32 to Compound i-30

A crude solution of triazole ethyl ester i-32 (7.5 g assay, 93% ee) in2-MeTHF was diluted with 250 mL of 2-Me-THF that was previouslysaturated with 1 M potassium phosphate buffer (pH 7.0). The resultingsolution was heated to 30° C., followed by the addition of Novozyme 435(15 g). The reaction was aged at 30° C. for 45 h.

The product ee was gradually decreased as the hydrolysis progressed. Thereaction significantly slowed down as the desired enantiomer wasconsumed. If the ee of the starting material (triazole ester) is lower,the reaction has to be stopped at a lower conversion before thehydrolysis of the undesired enantiomer becomes competitive. The ee ofthe product was determined by SFC analysis. The conversion can bedetermined by RPLC.

The reaction mixture was filtered to remove the immobilized enzyme, andthe enzyme was rinsed with 310 mL of buffer-saturated 2-Me-THF. Thecombined filtrate was assayed by HPLC. 5.74 g product (90% yield). >99%ee by SFC.

The solvent of the crude solution was switched from 2-Me-THF to IPA(total volume ˜115 mL) by distillation. Lithium acetate (2.44 g) and H₂O(9 mL) were added. The resulting slurry was aged at RT for 3 days andwas azeotropically distilled while feeding a total of 230 mL of IPA (40°C., 50 Torr) to remove acetic acid. 0.6 v/v % H₂O by KF. The slurry wascooled to RT and aged at RT for 4 h. The resulting solid was collectedby filtration, washed with IPA and suction-dried to afford the triazoleacid Li-salt i-30 as white solids (5.46 g). 92% isolated yield. >99.5%ee by SFC.

The enzyme can be recycled for re-use multiple times. The enzyme absorbsthe triazole acid product and needs to be rinsed thoroughly afterreaction to recover product. Adequate aging time for the Li-saltformation reaction was for from 12 hours to 3 days. The generatingacetic acid needed to be distilled off to drive the Li-salt formation tocompletion. The addition of H₂O was helpful to promote the Li-saltformation. LiOAc (weak base) was chosen in order to avoid the hydrolysisof the unreacted ester (low ee).

Using the Achiral HPLC method described in Step 1 (diluent: 5% MeCN/H₂O;product (i-30) peak gets broadened if prepared in different diluents),the retention times of i-30 and i-32 were about 5.7 min and 8.0 min,respectively.

Using the following Chiral HPLC method, the retention times of thedesired enantiomer (S) and undesired enantiomer (R) were about 4.4 minand 7.1 min, respectively.

Chiral SFC Method (Triazole Acid and Li-Salt)

Column: IC SFC, 250×4.6 mm 5 μm

Detector: UV 210 nm; Temp.: 35 C; Flow rate: 3.0 mL/min (200 bar);Injection: 10 μL;

Mobile phase A: CO₂; B: 25 mM isobutylamine in MeOH

Gradient program: Isocratic, 10% B for 12 min

Example 5 Preparation of Compound of Formula (II) from Compound i-11 andCompound i-30

To a mixture of pyrrolidine i-11 (2.16 g) and lithium triazole salt i-30(1.47 g) in water (11.6 mL) and IPA (6.48 mL) at 0-5° C. was added 5MHCl (3.52 mL) dropwise. The resulting solution was aged for additional30 min. EDCI (1.76 g) was charged in portions over 1 h while theinternal temperature was maintained 0-5° C. After 1-2 h age at 0-5C, >98% conversion was obtained. The mixture was aged overnight at RTand diluted with EtOAc (20 mL) and pH adjusted to 7-8 with NH₄OH (14 wt%, ˜4.5 mL) maintaining the internal temperature <5° C. The organicphase was separated and the aqueous phase was extracted with 10%IPA/EtOAc (10 mL).

The combined organic layer was washed with water (5 mL) andazeotropically solvent switched to IPA to a final volume of 17 mL. MTBE(23 mL) was added. After ˜10% of 0.87 ml of HOAc was added at RTdropwise, the batch was seeded. The slurry was aged at RT for 1 h toform a good seed bed. The rest of HOAc was added dropwise at RT over 2h. Then, the slurry was warmed to 40° C. and aged for 2 h before coolingto RT. After 2 h age at RT, the batch was filtered and washed with 30%IPA in MTBE (12 mL×2 displacement washes followed by a 12 mL slurrywash). The cake was vacuum oven dried at 40° C. to give 90% yield ofcompound of Formula (II) as an off-white solid.

Using the following HPLC method, the retention times of i-11 and Formula(II) were about 7.3 min and 8.4 min, respectively.

HPLC Method

Column: Restrek ultra II biphenyl, 4.6×1150 mm, 5.0 μm particle size;

Column Temp: 50° C.; Flow Rate: 1.5 mL/min; UV Detection: 220 nm;

Mobile Phase: A: 1% H₃PO₄ and 1% HClO₄; B: acetonitrile

Mobile Phase Program:

Time, min 0 3 12 13 13.01 15 A % 95 95 85 5 95 95 B % 5 5 15 95 5 5

While the invention has been described and illustrated with reference tocertain particular embodiments thereof, those skilled in the art willappreciate that various changes, modifications and substitutions can bemade therein without departing from the spirit and scope of theinvention. It is intended, therefore, that the invention be defined bythe scope of the claims which follow and that such claims be interpretedas broadly as is reasonable.

What is claimed is:
 1. A process of making compound I-11:

comprising: (a) reducing compound I-8:

in the presence of a catalyst to produce compound I-9:

(b) reacting compound I-9 with an acid to produce compound I-10:

and (c) reducing compound I-10 in the presence of a catalyst to producecompound I-11; wherein P¹ and P² are each independently selected fromthe group consisting of Ac, Bn, Boc, Bz, Cbz, DMPM, FMOC, Ns, Moz, andTs.
 2. The process of claim 1, wherein the catalyst in step (a) isselected from the group consisting of Pd, Raney Ni, Pt, PdCl₂, andPd(OH)₂; and reduction reaction is carried out in the presence ofhydrogen gas.
 3. The process of claim 1, wherein the acid in step (b) isselected from the group consisting of HCl, HBr, TFA, MeSO₃H, TfOH,H₂SO₄, para-toluenesulfonic acid, and RSO₃H wherein R is alkyl, aryl orsubstituted aryl.
 4. The process of claim 1, wherein the reduction ofstep (c) is carried out in the presence of HMDS and the catalyst used isselected from the group consisting of Pt on alumina, Pd on alumina,Pd/C, Pd(OH)₂—C, Raney Ni, Rh/C, Rh/Al, Pt/C, Ru/C and PtO₂.
 5. Theprocess of claim 1, further comprising reacting compound I-7;

with phosphonate compound A-4:

to produce compound I-8; wherein the reaction is carried out at atemperature of about 20 to 40° C. and in the presence of a solventselected from the group consisting of THF, MTBE, CH₂Cl₂, MeCN, tolueneand a mixture comprising two of the foregoing solvents; and wherein P¹and P² are each independently selected from the group consisting of Ac,Bn, Boc, Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts.
 6. The process of claim5, wherein the compound I-7 is in a solution, and the solutioncontaining compound I-7 is added to a solution containing compound A-4.7. The process of claim 5, further comprising oxidizing compound I-6:

with an oxidizing agent in the presence of a solvent and a catalyst toproduce compound I-7; wherein P¹ is selected from the group consistingof Ac, Bn, Boc, Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts.
 8. The process ofclaim 7, wherein: the solvent is selected from the group consisting ofTHF, MTBE, CH₂Cl₂, MeCN, toluene and a mixture comprising two of theforegoing solvents; the oxidizing agent is selected from the groupconsisting of NaOCl, NaClO₂, hydrogen peroxide, pyridine sulfurtrioxide, PCC, and DCC; and the catalyst is TEMPO or a TEMPO analogue.9. The process of claim 7, further comprising reacting compound I-5b:

with acetone and Boc₂O to produce compound I-6, wherein P¹ is Boc.
 10. Aprocess of producing compound I-11 comprising: (a) reacting, compoundI-5b;

with acetone and Boc₂O to produce compound I-6:

(b) oxidizing compound I-6 with an oxidizing agent in the presence of asolvent and a catalyst to produce compound I-7:

(c) reacting compound I-7 with phosphonate compound A-4:

to produce compound I-8:

wherein the reaction is carried out at a temperature of about 20 to 40°C. and in the presence of a solvent selected from the group consistingof THF, MTBE, CH₂Cl₂, MeCN, toluene and a mixture comprising two of theforegoing solvents; (d) reducing compound I-8 in the presence of acatalyst selected from the group consisting of Pd, Raney Ni, Pt, PdCl₂,and Pd(OH)₂ to produce compound I-9:

(e) reacting compound I-9 with an acid to produce compound I-10:

and (f) reducing compound I-10 in the presence of a catalyst to producecompound I-11:

wherein P¹ is Boc and P² is selected from the group consisting of Ac,Bn, Boc, Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts.
 11. A crystallineanhydrous form of compound of Formula I-11

characterized by XRPD by the following reflections with the d-spacing,Position [°2 Theta] d-spacing [Å] 17.8453 4.97 25.1979 3.53 20.1002 4.4223.9931 3.71 16.7073 5.31 25.5483 3.49 19.6576 4.52 13.8883 6.38 28.0863.18 20.6498 4.30.


12. A crystalline hemihydrate form of compound of Formula I-11

characterized by XRPD by the following reflections with the d-spacing,Position [°2 Theta] d-spacing [Å] 17.9681 4.94 17.8666 4.96 23.1905 3.8415.3049 5.79 19.7955 4.49 19.9483 4.45 23.1076 3.85 25.3415 3.51 16.08595.51 25.6746 3.47.